Mutations in DNAH1, Which Encodes an Inner Arm Heavy Chain Dynein, Lead to Male Infertility from Multiple Morphological Abnormalities of the Sperm ﬂagella

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Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm ﬂagella. Mariem Ben Khelifa, Charles Coutton, Raoudha Zouari, Thomas Karaouz`ene, John Rendu, Marie Bidart, Sandra Yassine, Virginie Pierre, Julie Delaroche, Sylviane Hennebicq, et al.

To cite this version: Mariem Ben Khelifa, Charles Coutton, Raoudha Zouari, Thomas Karaouz`ene,John Rendu, et al.. Mutations in DNAH1, which encodes an inner arm heavy chain dynein, lead to male infertility from multiple morphological abnormalities of the sperm ﬂagella.. American Journal of Human Genetics, Elsevier (Cell Press), 2014, 94 (1), pp.95-104. <10.1016/j.ajhg.2013.11.017>.

HAL Id: pasteur-01061012 https://hal-riip.archives-ouvertes.fr/pasteur-01061012 Submitted on 4 Sep 2014

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REPORT Mutations in DNAH1, which Encodes an Inner Arm Heavy Chain Dynein, Lead to Male Infertility from Multiple Morphological Abnormalities of the Sperm Flagella

Mariem Ben Khelifa,1,2,3,15 Charles Coutton,1,2,4,15 Raoudha Zouari,5 Thomas Karaouze`ne,1,2 John Rendu,1,6,7 Marie Bidart,1,7 Sandra Yassine,1,2 Virginie Pierre,1,2 Julie Delaroche,1,7 Sylviane Hennebicq,1,2,8 Didier Grunwald,1,7 Denise Escalier,9 Karine Pernet-Gallay,1,7 Pierre-Simon Jouk,10,11 Nicolas Thierry-Mieg,10 Aminata Toure´,12,13,14 Christophe Arnoult,1,2,16 and Pierre F. Ray1,2,6,16,*

Ten to fifteen percent of couples are confronted with infertility and a male factor is involved in approximately half the cases. A genetic etiology is likely in most cases yet only few genes have been formally correlated with male infertility. Homozygosity mapping was carried out on a cohort of 20 North African individuals, including 18 index cases, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF) including absent, short, coiled, bent, and irregular flagella. Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR, immunostaining, and electronic microscopy were carried out on samples from one of the subjects with a mutation located on a donor splice site. Neither the transcript nor the protein was observed in this individual, confirming the pathogenicity of this variant. A general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms was observed. Although DNAH1 is also expressed in other ciliated cells, infertility was the only symptom of primary ciliary dyskinesia observed in affected subjects, suggesting that DNAH1 function in cilium is not as critical as in sperm flagellum.

Male infertility affects more than 20 million men world- mainly composed of an intricate network of microtubules wide and represents a real health concern.1 It is a typical and dyneins. Sperm parameters have not been systemati- multifactorial disorder with a strong genetic basis and cally explored and are often only scarcely described additional etiological factors such as urogenital infections, in manuscripts investigating PCD-affected individuals. immunological or endocrine diseases, attack from reactive Although sperm flagella and motile cilia have a similar oxygen species (ROS), or perturbations from endocrine axonemal structure based on the presence of nine periph- disruptors. To date, despite substantial efforts made to eral microtubule doublets plus two central ones, they pre- identify genes specifically involved in male infertility by sent several differences that might explain why PCDs are many teams including ours,2,3 only a handful of genes not always associated with asthenozoospermia.4 We note have been formally correlated with human sperm defects. that no mutations in axonemal genes have been described Male infertility caused by impaired sperm motility (asthe- as being involved exclusively in infertility without also nozoospermia) is also often observed in men with primary inducing PCD. ciliary dyskinesia (PCD), a group of mainly autosomal- In the present study, we analyzed 20 subjects presenting recessive disorders caused by dysfunctions of motile cilia with asthenozoospermia resulting from a combination of leading primarily to respiratory infections and often to five morphological defects of the sperm flagella (absent, situs invertus. Recent research on PCD has been extremely short, bent, and coiled flagella and flagella of irregular prolific and allowed the identification and characterization width) without any of the other PCD-associated symp- of numerous proteins necessary for adequate axonemal toms. Similar phenotypes have been previously described molecular structure and assembly (Table S1 available and named ‘‘dysplasia of the fibrous sheath,’’ ‘‘short tails,’’ online). The axoneme is a highly evolutionarily conserved or ‘‘stump tails.’’5–15 We propose to call this syndrome structure found in motile cilia and in sperm flagella, ‘‘multiple morphological anomalies of the flagella

1Universite´ Joseph Fourier, Grenoble 38000, France; 2Laboratoire AGIM, CNRS FRE3405, Equipe ‘‘Andrologie et Geńe´tique,’’ La Tronche 38700, France; 3Laboratoire de geńomique Biome´dicale et Oncogeńe´tique, Institut Pasteur de Tunis, 1002 Tunis, Tunisie; 4CHU de Grenoble, Hoˆpital Couple Enfant, De´partement de Geńe´tique et Procreátion, Laboratoire de Geńe´tique Chromosomique, Grenoble 38000, France; 5Clinique des Jasmins, 23, Av. Louis BRAILLE, 1002 Tunis, Tunisia; 6CHU de Grenoble, Institut de Biologie et Pathologie, De´partement de Biochimie, Toxicologie et Pharmacologie (DBTP), UF de Biochimie et Geńe´tique Molećulaire, Grenoble 38000, France; 7INSERM, U836, Grenoble Institute of Neuroscience, La Tronche 38700, France; 8CHU de Grenoble, Hoˆpital Couple Enfant, De´partement de Geńe´tique et Procreátion, Laboratoire d’Aide a` la Procreátion – CECOS, Grenoble 38000, France; 9INSERM UMR_S933, Universite´ Pierre et Marie Curie (Paris 6), Paris 75012, France; 10Universite´ Joseph Fourier-Grenoble 1 / CNRS / TIMC- IMAG UMR 5525, Grenoble 38041, France; 11CHU de Grenoble, Hoˆpital Couple Enfant, De´partement de Geńe´tique et Procreátion, Service de Geńe´tique Clinique, Grenoble 38000, France; 12INSERM, U1016, Institut Cochin, Paris 75014, France; 13CNRS, UMR8104, Paris 75014, France; 14Universite´ Paris Descartes, Sorbonne Paris Cite´, Faculte´ de Me´decine, Paris 75014, France 15These authors contributed equally to this work 16These authors contributed equally to this work and are co-senior authors *Correspondence: [email protected] http://dx.doi.org/10.1016/j.ajhg.2013.11.017. Ó2014 by The American Society of Human Genetics. All rights reserved.

The American Journal of Human Genetics 94, 95–104, January 2, 2014 95 (MMAF),’’ a name that provides a more accurate descrip- the axoneme and that its depletion alters motility.17 The tion of this phenotype. We carried out a SNP whole- second subregion in chromosome 3 includes 28 genes genome scan on 20 individuals presenting with severe (Table S2). The dynein heavy chain 1 gene (DNAH1 MMAF. The study was approved by our local ethics com- [MIM 603332]) appeared as the best candidate gene mittee; all individuals gave their signed informed consent because it codes for an axonemal dynein heavy chain and national laws and regulations were respected. All indi- and is expressed in various tissues including testis.18 viduals originated from North Africa (11 Tunisians, 7 Furthermore, asthenozoospermia was described in mice Algerians, and 2 Libyans) and were treated in Tunis (Clin- lacking Dnahc1, the DNAH1 mouse ortholog (previously ique des Jasmins, Tunis, Tunisia) for primary infertility. named Mdhc7).19 Finally, among the ten genes located Twelve of the subjects were born from related parents, in the selected region of chromosome 20 (chr20: usually first cousins. None of the subjects were related to 33,572,687–34,070,415), only SPAG4 (MIM 603038) ap- one another apart from three individuals (P1–P3) who peared as a good candidate: it was described in rat to be were brothers. All subjects had normal somatic karyotypes. associated with the axoneme in elongating spermatids All sperm analyses were performed at least twice, in and epididymal sperm.20 We therefore decided to sequence accordance with the World Health Organization recom- KIF9 (RefSeq accession number NM_001134878.1), mendations.16 Subjects were recruited on the basis of the DNAH1 (RefSeq NM_015512.4), and SPAG4 (RefSeq identification of >5% of at least four of the aforemen- NM_003116.1). tioned flagellar morphological abnormalities (absent, We sequenced the 12 exons and the intron boundaries short, coiled, bent, and irregular flagella) (Table 1). All of SPAG4 in the 13 individuals homozygous at this locus, subjects presented with severe asthenozoospermia: 11 and the 19 exons and intron boundaries of KIF9 in the out of 20 subjects had no (0%) motility, 8 had sperm 15 relevant individuals. We did not identify any likely motility <10%, and one (P6) had 35% motility. Saliva pathogenic variants in these two genes. We then was obtained from all participants via Oragene DNA Self- sequenced the 78 exons and intron boundaries of Collection Kit (DNAgenotech) but only one subject (P3) DNAH1 in P3 (primer sequences available in Table S3). agreed to donate sperm and blood samples for research We identified one homozygous splicing mutation use. During their medical consultation for infertility, all (c.117881G>A) in intron 73. The same homozygous mu- subjects answered a health questionnaire focused on tation was identified in the two other brothers (Figure S2). PCD manifestations, and none indicated suffering from We then sequenced DNAH1 for the 17 remaining subjects. any of the other symptoms encountered in PCD. The same homozygous mutation (c.117881G>A) was Homozygosity mapping was carried out with 250K Sty1 identified in one additional individual (P17). We identified SNP mapping arrays (Affymetrix) on DNA extracted from three other homozygous variants: another splicing muta- the 20 studied subjects’ saliva samples. Common regions tion (c.5094þ1G>A) in individual P9, a homozygous no- of homozygosity were identified with the homoSNP soft- stop mutation disrupting the stop codon in exon 78 ware. After exclusion of the centromeric regions, we iden- (c.12796T>C [p.4266Glnext*21]) in individual P8, and a tified two regions located on chromosomes 3 and 20 with a homozygous missense variant in exon 23 in individual region of homozygosity > 1 Mb common to 10/20 P6 (c.3877G>A [p.Asp1293Asn]). The localization of the analyzed individuals (Figure S1). In addition, 4 and 9 sub- DNAH1 mutations is presented in Figure 1. If we consider jects presented with a stretch of homozygosity > 15 Mb only index cases, we identified 5 homozygous variants in overlapping chromosomes 20 and 3 regions, respectively. 18 unrelated individuals (28%). None of these variants All three brothers (P1–P3) were homozygous at the chro- were detected in our control cohort of 100 individuals of mosome 3 region, although only two of them were homo- North African origin. We note that the parents of the zygous at the chromosome 20 region. We excluded all subjects could not be analyzed to confirm the transmission other regions of homozygosity because they did not fulfil of the variants. We therefore cannot formally exclude the the following criteria: (1) more than eight individuals possibility that some of the identified variants may be including at least two of the brothers sharing a region of hemizygous with a deletion on the other allele. However, homozygosity > 1 Mb and (2) presence of a potential depending on its size, its position, and its effect on the candidate gene in the region according to its expression reading frame, a deleted allele would be at least as delete- profile and/or presumed function. Finer analysis of the rious as the identified variants. chromosome 3 region showed that 15 individuals were To evaluate the association of the variants with the homozygous for two smaller subregions located at chr3: pathology, we compared their frequency in our cohort 46,745,396–47,606,570 and chr3: 52,111,974–53,028,375 with that in the Exome Variant Server (EVS) database. At (UCSC Genome Browser human reference genome build the four genomic positions of interest, the EVS data are hg17, Figure S1). Sixteen genes are annotated in the first of sufficient coverage to provide genotype calls for at least subregion (Table S2), among which only one gene (KIF9 6,200 individuals, corresponding to 12,400 alleles. There [MIM 607910]) appeared as a good candidate; indeed, were no variant nucleotides identified at positions studies in the protist Trypanosoma brucei showed that c.5094þ1, c.117881, or c.12796, and only one A allele kif9A (the mouse ortholog of human KIF9) is located in was identified out of 12,460 alleles at position c.3877.

96 The American Journal of Human Genetics 94, 95–104, January 2, 2014 Table 1. Semen Parameters of the 20 Subjects and the 7 Subjects Carrying DNAH1 Homozygous Variants Average of Semen Parameters 20 Subjectsa P1 P2 P3 P6 P8 P9 P17

DNAH1 mutations c.117881G>A c.117881G>A c.117881G>A c.3877G>A c.12796 T>C c.5094þ1G>A c.117881G>A (p.Gly3930Alafs*120) (p.Gly3930Alafs*120) (p.Gly3930Alafs*120) (p.Asp1293Asn) (p.4266Glnext*21) (p.Leu1700Serfs72) (p.Gly3930Alafs*120)

Consanguinity yes yes yes no yes yes no

Origin of the Tunisia Tunisia Tunisia Algeria Algeria Algeria Tunisia subject

Sperm volume (ml) 3.2 (1–5.5) 5 2.5 2 5 4.5 3.5 2.5

Sperm concentration 22 (0–59) 45 0 2.8 57 11 53 31 6

h mrcnJunlo ua Genetics Human of Journal American The 10 /ml

Motility (AþB) 1 hr 2.5 (0–35) 0 NA 2 35 0 0.5 0

Vitality 44 (6–73) 22 NA NA 73 61 48 NA

Normal spermatozoa 0.35 (0–6) 0 NA 0 6 0 0 0

Absent ﬂagella 30 (8–46) 34 þ 34 þþ16 30

Short ﬂagella 44 (16–70) 38 þ 44 þþ70 20

Coiled ﬂagella 13 (2–32) 14 NA 14 þþ12 32

Angulation 12 (2–19) 4 NA 2 þþ 86

Flagella of irregular 55 (16–92) 48 NA 50 þþ54 16 caliber

Multiple anomalies 2.9 (1.9–3.9) 3.1 NA 2.4 NA NA 3 2.6 94 index 514 aur ,2014 2, January 95–104, ,

Values are expressed in percents, unless specified otherwise. Abbreviations are as follows: NA, not available; plus sign, anomalies reported (>5%) but not accurately quantified. aValues are expressed as the mean with the lower and higher values in parentheses. 97 Figure 1. Location of DNAH1 Mutations in the Intron-Exon Structure and in the Protein Representation of DNAH1 (A) DNAH1 genomic structure. (B) DNAH1 domain map showing the location of the four identified mutations. The red boxes indicate the six known AAA- ATPase domains (AAA 1 to 6) as detected by homology (Uniprot server). The microtubule-binding domain (MBT) lies between AAA4 and AAA5. The N-terminal part of the protein binds to the intermediate, light-intermediate dynein chains. The position of the stalk and the microtubule- binding domain (MTB) are indicated.

We performed Fisher exact tests (with fisher.test in R) to p.4266Glnext*21 found in individual P8 abolishes the stop evaluate whether each observed SNV was statistically over- codon in exon 78, leading to the addition of 21 codons represented in our cohort of 18 unrelated individuals, at the 30 end of the coding sequence. The role of the compared to EVS. All four individual SNVs are significantly C-terminal domain is uncertain, but based on the enriched (p values: 5.9 3 10 11 for c.117881G>A, 8.1 3 D. discoideum structures, it may participate in long-range 10 6 for c.5094þ1G>A and c.12796T>C, and 2.4 3 10 5 allosteric communication between microtubule-binding for c.3877G>A). We then investigated whether DNAH1 and ATPase regions.22,23 The addition of 21 extra amino as a whole was significantly enriched in damaging SNVs acids to this region is likely to disrupt these interactions. in our cohort. Overall, EVS contains five nonsense and The c.117881G>A variant identified in four subjects two splice-site SNVs, all observed heterozygously in a total (P1–P3 and P17) affects the final G nucleotide of DNAH1 of ten individuals. By using the coverage data available on intron 73, one of the consensus splice acceptor nucleo- the EVS website, we find that 20,737 positions covering tides. The resulting abnormal splicing is predicted to recog- the DNAH1 exons and intron boundaries had sufficient nize a new CG acceptor site located just one nucleotide sequence coverage to be genotyped in 6,189 individuals further, thus shifting the reading frame and inducing a on average. By contrast, counting only the two splice-site premature stop codon (p.Gly3930Alafs*120). As could be mutations as damaging, we observe 6 damaging alleles expected, P1–P3 share a common haplotype (Table S4). among 36 in our cohort: this represents a highly signifi- P17 also shares a common haplotype of 30 SNPs with cant enrichment (Fisher exact test, p value: 3 3 10 12). P1–P3, suggesting a founder effect for this mutation. To Furthermore, we note that there were no homozygous assess the functional impact of the DNAH1 splice acceptor damaging variants observed in the 6,189 EVS individuals site mutation c.117881G>A, we studied mRNA products compared to 3 in our cohort of 18 (Fisher exact test, isolated from control and P3 lymphocytes (primer se- p value: 3 3 10 8). Altogether we believe that these genetic quences available in Table S5). RT-PCR of P3’s samples results convincingly demonstrate that mutations in yielded no product despite repeated attempts, whereas DNAH1 are associated with MMAF. the three amplification attempts from control lympho- The c.5094þ1G>A variant found in individual P9 affects cytes yielded the expected product (Figure 2A). RT-PCR tar- DNAH1 intron 31 consensus donor splice site. The geting GAPDH (MIM 138400) and RPLP0 (MIM 180510) abnormal splicing is predicted to cause the prolongation confirmed the integrity of P3’s RNA (Figure 2B). This sug- of exon 31 until the introduction of a nonsense codon gests a specific degradation of the mutant DNAH1 tran- (p.Leu1700Serfs72). The position of the next donor site scripts by NMD. To further validate the pathogenicity of was predicted by ‘‘Splice Site Prediction by Neural this variant, we analyzed DNAH1 localization in sperm Network.’’ Unfortunately we could not obtain any leuko- from P3 by immunofluorescence and the ultrastucture of cytes from this subject to validate this prediction and the flagella by electron microscopy. In control individuals, observe whether this variant also led to nonsense-medi- DNAH1 antisera decorated the full length of the sperm fla- ated mRNA decay (NMD). Because of the location of the gellum (Figure 2C), suggesting a putative role in the teth- variant on a consensus splice site and the unambiguous ering of the inner dynein arms along the entire axoneme. predictions of splice prediction software, we did not syn- In contrast, in sperm from individual P3 carrying the thesize a minigene to verify the effect of this variant c.117881G>A mutation, DNAH1 immunostaining was in vitro. A missense change, p.Asp1293Asn, was identified absent, confirming that the splicing defect results in the in P6. Interestingly, the Asp1293 amino acid is well degradation of the transcripts by NMD (Figure 2D). We conserved across species (Figure S3). This missense change next tested the integrity of the outer and inner dynein is also predicted to be possibly damaging by SIFT and Poly- arms by using antibodies directed against DNALI1 and Phen-2, two prediction softwares for nonsynonymous DNAI2, two well-established diagnostic markers of the in- SNPs. It affects the N-ter of the protein (Figure 1B), known ner and outer dynein arms, respectively. Staining with to be important for the structure of dynein arms.21 Variant DNALI1 was strongly reduced in the sperm of individual

98 The American Journal of Human Genetics 94, 95–104, January 2, 2014 Figure 2. Analysis of P3 Carrying the c.117881G>A Variant Evidencing DNAH1 mRNA Decay by RT-PCR and the Absence of DNAH1 in Sperm by Immunolocalization (A) RT-PCR analyses of subject P3 (c.117881G>A homozygote) and control individuals from the general population (C1–C3). Electro- phoresis showing the RT-PCR amplification of DNAH1 exons 30–32, 64–66, 73–75. C1, C2, and C3 yield a normal fragment of 228, 293, and 241 bp, whereas subject P3 shows no amplification. There is no amplification from the RT-negative blank control (column B). (B) Electrophoresis showing the amplification of the same cDNAs with GAPDH and RPL0 primers. Bands of equivalent intensity are obtained from all samples including P3. Reverse transcription was carried out with 500 ng of extracted RNA and oligo dT priming. Two microliters of the obtained cDNA mix was used for the subsequent PCR. PCR amplification was carried out with three couples of primers located in exons 30–32, 64–66, and 73–75 of DNAH1 at an elongation temperature of 57C (40 cycles), in parallel to amplification of the same samples with the control housekeeping GAPDH and RPL0, respectively, at an elongation temperature of 60C (35 cycles). RT-PCR primers are listed in Table S4. (C and D) Immunofluorescence staining of human spermatozoa with DNAH1 antibodies (green) and DNAI2 (red). DNAH1 is observed throughout the flagellum in control sperm, whereas it is absent from P3’s sperm. In both control and P3 sperm, ODA is present as wit- nessed by the immunostaining of DNAI2. (E and F) Immunofluorescence staining of human spermatozoa with DNALI1 antibodies (green) and DNAI2 (red). DNALI1, a marker of IDA, is localized throughout flagella in control sperm, whereas it is strongly reduced in sperm from P3. No difference is noticed in the coimmunostaining of DNAI2. Sperm were counterstained with Hoechst 33342 (blue) as nuclei marker. White scale bars represent 5 mm. Sperm cells were washed in phosphate-buffered saline (PBS), fixed in 4% PFA for 2 min at room temperature (RT), and washed twice in PBS. Fixed spermatozoa were allowed to air-dry on poly-L-lysine coated slides followed by permeabilization with 0.5% Triton X-100. Samples were then blocked with (PBS)/1% bovine serum albumin (BSA)/2% normal goat serum (NGS) for 30 min at RT. Slides were incu- bated with the primary antibodies 2 hr followed by an incubation with the secondary antibodies for 45 min at RT and mounting in Dako mounting medium (Dako). Appropriate controls were performed, omitting the primary antibodies. Polyclonal mouse DNALI1 and monoclonal mouse DNAI2 were purchased from Abcam (UK) and Abnova Corporation (Taiwan), respectively. Polyclonal DNAH1 antibodies were purchased from Prestige Antibodies (Sigma-Aldrich). Monoclonal mouse anti-acetylated-a-tubulin were purchased from Sigma-Aldrich. Highly cross-adsorbed secondary antibodies (Alexa Fluor 488 and Alexa Fluor 546) were obtained from Molecular Probes (Invitrogen).

P3, suggesting that inner arms were mostly absent in this confirming the complete disorganization of the IDA. More- individual (Figure 2F). On the other hand, the antibodies over, approximately one third of the microtubule doublets directed against DNAI2 stained the sperm flagella in both were malformed or absent in the observed sections. control and individual P3, suggesting that the outer Furthermore, the central singlet of microtubules was dynein arms were not affected by the absence of DNAH1 missing (9þ0) in 47% of these sections. The fibrous sheath (Figures 2E and 2F). In order to confirm that the inner was also strongly disorganized in 90% of the sections arms were disorganized, we studied the ultrastructure of (Figure 3). individual P3’s sperm by transmission electron microscopy After complete DNA sequencing of DNAH1, we identi- (TEM) (Figure 3). We could observe 40 doublets of microtu- fied two variants altering a consensus splice site, highly bules in cross sections presenting a sufficient quality to likely to have a damaging effect, in 3 out of 18 unrelated observe the dynein arms. Fifteen outer dynein arms individuals. The predicted effect of the other two identified (ODA) and only 4 inner dynein arms (IDA) were observed, variants is not as clear but the addition of 21 residues

The American Journal of Human Genetics 94, 95–104, January 2, 2014 99 Figure 3. Electron Microscopy Analysis of Spermatozoa from P3, Carrying the c.117881G>A Variant, Reveals Numerous Ultrastruc- tural Defects (A) EM cross-section of a flagellum from a control individual sperm sample. (B) Cross-section from the individual P3 sample showing numerous defects: lack of IDA (red arrow) and axonemal disorganization with mislocalized peripheral doublets (green arrows) associated with a displacement of the central pair (blue asterisk). (C) Cross-section from individual P3 sperm flagellum showing a complete absence of the central pair. (D) Cross-section from individual P3 sperm flagellum showing supernumerary dense fibers with absence of mitochondrion on the right side of the mid-piece. (E and F) Drawings describing the normal sperm axoneme ultrastructure with their different components (E) and different defects observed in DNAH1 mutated subjects (F). Sperm cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) during 2 hr at room temperature. Details of transmission electron microscopy technique were detailed previously.3 caused by the stop-loss variant is probably pathogenic. We can therefore expect that individuals with intermediate Interestingly, P1, P2, P3, P8, P9, and P17—who have asthenozoospermia and low levels of morphological inherited the variants predicted to have most severe anomalies could also harbor homozygous or compound effects—present 0% morphologically normal spermatozoa heterozygous DNAH1 mutations of moderate severity. with a motility <2%, in contrast to P6, carrying the The data we present here are consistent with the pheno- p.Asp1293Asn variant, who presents a milder phenotype type described for Dnahc1 knockout (KO) mice (the ortho- with 35% motility and 6% morphologically normal sper- log of DNAH1), which display asthenozoospermia and matozoa (Table 1). These data therefore suggest that the male infertility.19 In this model, however, no structural c.3877G>A variant might be a hypomorphic allele, which defects of the axoneme were observed either by optical or is consistent with a single amino acid substitution in a by transmission electronic microscopy.19 This contrasts large protein. Unfortunately we could not obtain any addi- with the strong axonemal disorganization we observed in tional biological material from the other mutated subjects sperm from P3 carrying the homozygous c.117881G>A and in particular from P6 and could not assess the effects of mutation, where the inner dynein arms and the central the other variants on protein expression/localization and pair of microtubules were mostly absent. In the Dnahc1 on the ultrastructure of the flagella. In addition, no muta- KO, however, the authors describe that the targeted tions were identified in DNAH1 in 13 subjects, suggesting deletion did not lead to a complete disruption of the that MMAF is genetically heterogeneous. We are currently gene and resulted in a truncated protein with a preserved sequencing the exomes of these 13 subjects in order to N terminus.19 Because the N-terminal part of the DyHCs identify other genes involved in MMAF. We note that plays a crucial role in the assembly and stabilization of with the exception of P6, who carried a missense mutation the inner dynein arms, as shown in Chlamydomonas and presented a milder form of the pathology, we included mutants,24 it is likely that the formation of the base of here only individuals with the most severe phenotypes. the inner dynein arm is preserved and that the described

100 The American Journal of Human Genetics 94, 95–104, January 2, 2014 DNAH1 and DNAH12 expression is rather atypical, because it is almost ubiquitous (EST profile viewer and GeneHub- GEPIS). We therefore note that not only are DNAH1 and DNAH12 closest to one another from a phylogenetic point of view, but they also share a broad expression pattern. We therefore believe that DNAH12 is the most likely candidate for a potential functional compensation of DNAH1 in ciliated cells. In addition and/or alternatively to this compensation, we cannot exclude the possibility that some of the affected individuals might retain expression of DNAH1 in the ciliated cells of the trachea, although this is not expected for the most severe variants identified. Alterna- tively, we cannot exclude a reduction of ciliary beats, Figure 4. Relative mRNA Expression of DNAH1, DNAH3, DNAH7, which could lead to a small decrease of cilia function in DNAH12 and in Testis and in Tracheal Cells respiratory epithelium or in other ciliated tissues without Expression of DNAH1 mRNA (in green) in testis is significantly higher (8-fold) than in tracheal cells (in blue). Tracheal cell expres- pathological consequences, or at least none that have sion is set to 1. Expression of DNAH3 and DNAH7 mRNA are signif- been noticed by the affected men themselves. We could icantly lower in testis than in tracheal cells, whereas DNAH12 not obtain nasal brushings or curette biopsies from expression is not significantly different. Data are presented as 5 affected individuals and therefore cannot formally exclude mean standard deviation of three independent quantitative DNAH1 real-time PCR experiments. Statistical tests (paired t test) with a this possibility. Future work on mutated subjects two-tailed p value % 0.05 were considered as significant (*). Hu- should include a thorough analysis of PCD symptoms man testis and trachea cDNA were obtained from Amsbio (Abing- including nasal nitric oxide measurements, video micro- don). mRNA expression were assessed by qPCR with a Biorad CFX9 scopy, and transcription electron microscopy. This would (Biorad). PCR primers used to amplify DNAH1 and three other inner dynein arm heavy chain genes (DNAH3, DNAH7, and provide valuable information regarding the role of DNAH12) and the reference gene ACTB are listed in Table S5. DNAH1 in ciliated cells as well as indicate whether The PCR cycle was as follows: 10 min 95 C, 1 cycle; 10 s 95 C, mutated men might be at risk of developing PCD symp- þ 30 s 58 C fluorescence acquisition, 55 cycles. Analysis was per- toms, perhaps as late onset. formed with Biorad software CFX Manager v.3.0, with advanced relative quantification mode. Values for each gene were normal- Inner dynein arms are organized in seven molecular ized to expression level of beta-actin gene (ACTB) via the 2- complexes, viewed in electronic microscopy as globular DDCT method.25 The 2-DDCT value was set at 0 in tracheal cells, heads arranged in 3-2-2 groups and corresponding to three resulting in an arbitrary expression of 1. different types of inner arms (IDA1 to IDA3, see Figure 5). In Dnahc1 KO mice, electron microscopy studies indicated KO animals could ensure correct axonemal biogenesis and that one head of the IDA3 was missing, leading to a 3-2-1 organization. Alternatively, it is possible that the DNAH1 globular head arrangement, suggesting that DNAH1 is a role in axonemal structure is not as central in mouse as it component of IDA3. Radial spokes are present on microtu- is in human. bule doublets and interact with the inner arms. They allow Apart from infertility, none of the 20 individuals a connection between external doublets of the microtu- declared suffering from any of the principal PCD symp- bules and the two central microtubules. They are multipro- toms such as an impairment of the respiratory functions. tein complexes of more than 20 proteins. In mammals, This suggests that DNAH1 function in cilia is probably there are three different radial spokes (RS1, RS2, and RS3) compensated by other HC dyneins. Previous phylogenetic binding tightly to the inner arm bases of different IDAs. studies indicate that DNAH3 (MIM 603334), DNAH7 (MIM Among the different proteins involved in axonemal forma- 610061), and DNAH12 (MIM 603340) are close paralogs to tion and organization, only mutations in CCDC39 (MIM DNAH1, DNAH12 being the closest.18 We therefore 613798) and CCDC40 (MIM 613799) lead to a disorganiza- measured the relative expression of these four IDA heavy tion of the axonemal structure, a phenotype similar to chains to assess whether the expression of these proteins what we observe in subjects with DNAH1 mutations. could compensate the absence of DNAH1 in other ciliated CDCC39 and CDCC40 control the assembly of the dynein tissues. By using qPCR (primer sequences available in Table regulatory complex (DRC), a major regulatory node inter- S6), we showed that DNAH1 is expressed at a much higher acting with numerous axonemal structures.27–29 In the level (8-fold) in the testis compared to the trachea absence of DRC, RS2 anchoring is weakened, leading to (Figure 4). Conversely, the other IDA heavy chains are ex- the displacement or the absence of the central pair and pressed at higher levels (DNAH3, DNAH7) or at a similar the mislocalization of the peripheral doublets. Interest- level (DNAH12) in control tracheal cells as in testis ingly, in T. thermophila, the RS3 stalk is directly connected (Figure 4). Data available from public expression databases to the dynein d/a tail through an arc-like structure show a similar expression pattern to what was observed (Figure 5).26 It can therefore be speculated that the absence here. Moreover, these data show that whereas DNAH3 of DNAH1 removes the anchoring site of the radial spoke and DNAH7 expression is restricted to ciliated cells, 3. As a consequence, the attachment of the two central

The American Journal of Human Genetics 94, 95–104, January 2, 2014 101 p28 remain located in the flagella. It can thus be speculated that there are several pathways for the preassembly and/or the targeting of the different mammalian IDAs, thus explaining the absence of axonemal microtubule disorganization in subjects presenting with mutations in DNAAF1, DNAAF2, DNAAF3, and LRRC6. Here we observed that DNALI1 immunostaining was strongly reduced along the whole flagellum (Figure 2E), suggesting that DNALI1 may be located mainly in IDA3.40 In agreement with this result, p28, the Chlamydomonas ortholog of DNAIL1, is associated with the inner dynein arm located in IDA3. Interestingly, DNALI1 has an expression pattern similar to DNAH1: it presents a predominant testis expression and also a remarkable expression in nonciliated cells.41 Altogether, these facts suggest a close molecular partnership between DNAH1 and DNALI1. Mutations affecting axonemal components and/or axoneme assembly often result in PCD, which frequently includes a male infertility phenotype. In this study we describe that mutations in DNAH1, which codes for an Figure 5. Proposed Schematic Model for the Location and the axonemal component, leads to male infertility only with Function of the Inner Arm Heavy Chain DNAH1 in the Axoneme no other apparent PCD-associated syndromes. Our data of Human Sperm Flagellum indicate that DNAH1 is required in spermatozoa for the (A) Simplified representation showing a cross-sectional view of one microtubule doublet of an axoneme surrounding the central formation of the inner dynein arms and that its absence pair complex; the viewing is from the flagellar base. Light gray, is deleterious for the organization and biogenesis of the central pair complex; light blue, outer doublets; dark blue, outer axoneme. Overall our data confirm that despite close struc- arm dyneins (OAD); dark pink, inner arm dynein heavy chain tural similarities, sperm flagella and cilia present important DNAH1; dark gray, radial spoke 3. (B) Longitudinal view illustrating the approximate localization on divergences in axonemal organization and biogenesis. the outer doublet A-tubule of the various dyneins and regulatory structures within a single 96 nm axonemal repeat. RS3 stalk is directly connected to the DNAH1 tail through an arc-like struc- Supplemental Data ture. DNAH1 may therefore stabilize the RS3.26 Light pink, inner arm dyneins (IAD); yellow, IC/LC, intermediate chain/light chain; Supplemental Data include three figures and six tables and can be orange, nexin-dynein regulatory complex (N-DRC); green, modi- found with this article online at http://www.cell.com/AJHG/. fier of inner arms (MIA) complex; blue, calmodulin- and spoke- associated complex (CSC); purple, distal protrusion (DP). Acknowledgments singlet microtubules should be weakened. This scheme is This work was supported by the research grant ICG2I funded by in agreement with our data that show 47% of 9þ0 axo- the program GENOPAT 2009 from the French Research Agency nemes (Figure 3). (ANR). The funders had no role in study design, data collection As illustrated in Table S1, not all PCDs are associated and analysis, decision to publish, or preparation of the manu- script. We are grateful to Fre´de´ric Plewniak, IGBMC, Strasbourg, with an infertility phenotype. For instance, subjects with who developed the homoSNP software used in this study (the soft- mutations in CCDC114 (MIM 615038)30,31 and DNAH11 ware is available on request to [email protected]). (MIM 603339)4 are fully fertile and could procreate sponta- DNAAF2 neously whereas subjects with (MIM 612517), Received: July 19, 2013 DNAH5 DNAI1 HYDIN (MIM 603335), (MIM 604366), or Accepted: November 18, 2013 4,32–34 (MIM 610812) present complete sperm immobility. Published: December 19, 2013 Also, mutations in genes involved in the preassembly of the dynein arms DNAAF1 (MIM 611390), DNAAF2 (MIM 612517), DNAAF3 (MIM 614566),32,35–37 and LRRC6 Web Resources 38 (MIM 614930), described to induce IDA loss, did not pre- The URLs for data presented herein are as follows: sent axonemal microtubule disorganization. Consistently, 1000 Genomes, http://browser.1000genomes.org IDAs are not or are only partially affected in deficient Berkeley Drosophila Genome Project NNSplice 0.9, http://www. Chlamydomonas ODA7 DNAAF1 ktu/pf13 mutants for ( ), fruitfly.org/seq_tools/splice.html DNAAF2 pf22 DNAAF3 ODA7 ( ), and ( ): in mutants, inner dbSNP v.137, http://www.ncbi.nlm.nih.gov/projects/SNP/ arms are not affected, in ktu/pf13 mutants only IDA dynein snp_summary.cgi c is missing, and in pf22 mutants IDA dynein b and c are GeneHub-GEPIS, http://research-public.gene.com/Research/ absent.27,29,36,37,39 Most importantly, IDA dynein f and genentech/genehub-gepis/

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